The invention relates to novel nucleic acid molecules encoding proteins involved in the synthesis and assembly of core lipopolysaccharide of P. aeruginosa, the novel proteins encoded by the nucleic acid molecules; and, uses of the proteins and nucleic acid molecules.
Gram negative bacterial infections account for a significant number of hospital-acquired infections. The majority of hospital-acquired infections are due to gram negative organisms such as Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Gram negative infections are particularly common among individuals receiving chemotherapy, and immunocompromised individuals. These individuals often develop resistance to antibiotics over the long course of the infection making conventional treatment difficult.
Many virulence factors have been identified in the pathogenesis of gram negative bacteria, including lipopolysaccharide. The lipolypolysaccharide of gram negative bacteria is composed of O-antigen, usually tri- or tetrasaccharide repeating units, which is immunodominant and responsible for serotype specificity. The O-antigen is attached to a core oligosaccharide composed of hexoses and octoses, which is itself attached to lipid A (endotoxin) embedded in the cell membrane. The core lipopolysaccharide structure, particularly the inner core region, appears to be widely shared among diverse gram negative bacterial genera.
Genes involved in the biosynthesis of core oligosaccharides have been cloned and characterized from several bacterial species, including Escherichia coli, ( Parker et al., J. Bacteriol. 174, 93-0934, 1992; Genbank Accession No. M8O599, M86935), Salmonella typhimurium (Klena et al., J. Bacteriol 175(5) 1524-1527, 1993; Genbank Accession No. S56361), and Haemophilus influenzae (High N. J et al., Mol. Microbiol. 9(6) 1275-1282, 1993; Genbank Accession No. L19441).
The present inventors have characterized a gene cluster involved in the synthesis and assembly of core lipopolysaccharide of P. aeruginosa. The gene cluster is also known as and referred to herein as the waa (or rfa) gene cluster, and the proteins encoded by the genes are referred to herein as Waa (or Rfa) proteins.
The waa gene cluster contains the genes waaF, waaC, waaG and waaP. The arrangement of the genes in the waa gene cluster is shown in FIG. 2, and their role in the biosynthesis of the lipopolysaccharide core structure of P. aeuroginosa serotypes O5 and O6 is shown in FIG. 1.
The identification and sequencing of the genes and proteins in the waa gene cluster permits the identification of substances which affect core lipopolysaccharide synthesis or assembly in P. aeruginosa. These substances may be useful in inhibiting core lipopolysaccharide synthesis or assembly rendering the microorganisms more susceptible to attack by host defence mechanisms.
Broadly stated the present invention relates to an isolated P. aeruginosa waa gene cluster comprising the genes waaF, waaC, waaG, and waaP involved in the synthesis, and assembly of core lipopolysaccharide in P. aeruginosa. 
The present invention also relates to nucleic acid molecules encoding WaaF, WaaC, WaaG and WaaP proteins.
The invention also contemplates a nucleic acid molecule comprising a sequence encoding a truncation of a protein of the invention, an analog, or a homolog of a protein of the invention, or a truncation thereof.
The nucleic acid molecules of the invention may be inserted into an appropriate expression vector, i.e. a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Accordingly, recombinant expression vectors adapted for transformation of a host cell may be constructed which comprise a nucleic acid molecule of the invention and one or more transcription and translation elements operatively linked to the nucleic acid molecule.
The recombinant expression vector may be used to prepare transformed host cells expressing a protein of the invention. Therefore, the invention further provides host cells containing a recombinant molecule of the invention.
The invention further provides a method for preparing a protein of the invention utilizing the purified and isolated nudeic acid molecules of the invention. In an embodiment a method for preparing a protein of the invention is provided comprising (a) transferring a recombinant expression vector of the invention into a host cell; (b) selecting transformed host cells from untransformed host cells; (c) culturing a selected transformed host cell under conditions which allow expression of the protein; and (d) isolating the protein.
The invention further broadly contemplates an isolated protein characterized in that it has part or all of the primary structural conformation (ie. continuous sequence of amino acid residues) of a novel protein encoded by a gene of the waa gene cluster of the invention. In an embodiment of the invention, a purified protein is provided which has the amino acid sequence as shown in FIG. 4, FIG. 6, FIG. 7, or FIG. 9. The invention also includes truncations of the protein and analogs, homologs, and isoforms of the protein and truncations thereof.
The proteins of the invention may be conjugated with other molecules, such as proteins, to prepare fusion proteins. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins.
The nucleic acid molecules of the invention allow those skilled in the art to construct nucleotide probes for use in the detection of nucleotide sequences in samples such as biological (e.g clinical specimens), food, or environmental samples. The nucleotide probes may also be used to detect nucleotide sequences that encode proteins related to or analogous to the proteins of the invention.
Accordingly, the invention provides a method for detecting the presence of a nudeic acid molecule having a sequence encoding a protein of the invention, comprising contacting the sample with a nucleotide probe which hybridizes with the nucleic acid molecule, to form a hybridization product under conditions which permit the formation of the hybridization product, and assaying for the hybridization product.
The invention further provides a kit for detecting the presence of a nucleic acid molecule having a sequence encoding a protein of the invention, comprising a nudeotide probe which hybridizes with the nucleic acid molecule, reagents required for hybridization of the nucleotide probe with the nucleic acid molecule, and directions for its use.
The nucleic acid molecules of the invention also permit the identification and isolation, or synthesis, of nucleotide sequences which may be used as primers to amplify a nucleic acid molecule of the invention, for example in the polymerase chain reaction (P CR).
Accordingly, the invention relates to a method of determining the presence of a nucleic acid molecule having a sequence encoding a protein of the invention in a sample, comprising treating the sample with primers which are capable of amplifying the nucleic acid molecule in an amplification reaction, preferably in a polymerase chain reaction, to form amplified sequences, under conditions which permit the formation of amplified sequences, and, assaying for amplified sequences.
The invention further relates to a kit for determining the presence of a nucleic acid molecule having a sequence encoding a protein of the invention in a sample, comprising primers which are capable of amplifying the nucleic acid molecule in an amplification reaction, preferably a polymerase chain reaction, to form amplified sequences, reagents required for amplifying the nudeic acid molecule thereof in the amplification reaction, means for assaying the amplified sequences, and directions for its use.
The invention also relates to an antibody specific for an epitope of a protein of the invention or a part thereof, and methods for preparing the antibodies. Antibodies specific for a protein encoded by a waa gene of the invention can be used to detect P. aeruginosa of all serotypes in a sample.
Therefore, the invention also relates to a method for detecting P. aeruginosa of all serotypes in a sample comprising contacting a sample with an antibody specific for an epitope of a protein encoded by a waa gene of the invention which antibody is capable of being detected after it becomes bound to a protein in the sample, and assaying for antibody bound to protein in the sample, or unreacted antibody.
A kit for detecting P. aeruginosa serotypes in a sample comprising an antibody of the invention, preferably a monoclonal antibody and directions for its use is also provided. The kit may also contain reagents which are required for binding of the antibody to the protein in the sample.
As discussed above, the identification and sequencing of genes in the waa gene cluster in P. aeruginosa permits the identification of substances which affect the activity of the proteins encoded by the genes in the duster, or the expression of the proteins, thereby affecting core lipopolysaccharide synthesis or assembly. These substances may be useful in rendering the microorganisms more susceptible to attack by host defence mechanisms. Accordingly, the invention provides a method for assaying for a substance that affects one or both of P. aeruginosa core lipopolysaccharide synthesis or assembly comprising mixing a protein or nucleic acid molecule of the invention with a test substance which is suspected of affecting P. aeruginosa core lipopolysaccharide synthesis or assembly, and determining the effect of the substance by comparing to a control.
Substances that inhibit the synthesis or assembly of core lipopolysaccharides may be useful in treating or preventing bacterial infections by rendering the bacteria more susceptible to attack by host defense mechanisms. Accordingly, the present invention also provides a method for preventing or treating the bacterial infection comprising administering an effective amount of a substance that inhibits the synthesis or assembly of core lipopolysaccharides. In one embodiment, the substance inhibits the activity of one or more Waa proteins of the invention. Such substances include antibodies to the Waa proteins or other substances that bind the Waa proteins. In another embodiment, the substances may inhibit the expression of one or more waa genes. Such substances include antisense oligonucleotides that bind one or more waa genes or other substances that bind the nucleic acid sequences of the invention.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.